.beta.-thalassemias and sickle-cell anemia are human genetic disorders of the .beta.-globin gene, with severe clinical manifestations in homozygotes. At the present time, allogeneic bone marrow transplantation represents the best available possibility of cure for these patients. Unfortunately, few of them are able to locate a normal HLA-matched donor and even if a matched HLA donor were available, many would face severe complications of the bone marrow transplantation procedure such as graft versus host disease (Parkman, R., Science 232:1373-1378 (1986)). For these reasons, gene therapy using genetically modified autologous totipotent hematopoietic stem cells (THSC) is an attractive alternative to allogeneic bone marrow transplantation. Because gene targeting by homologous recombination is not yet technically possible in THSC, the most realistic strategy is to obtain stable integration of a normal human .beta.-globin gene and its cis-acting regulatory elements into the THSC genome. This can be achieved by retrovirus-mediated gene transfer, an efficient gene transfer technique applicable to these cells (Fraser et al., Blood, 76:1071-1076 (1991)).
Gene transfer experiments have previously shown that the proximal cis-acting elements of the human .beta.-globin gene are insufficient for gene therapy applications because they provide a very low, integration site-dependent expression of the human .beta.-globin transgene (less than 1 to 5% of human .beta.-globin/murine.beta..sub.maj -globin mRNA ratio) (Cone et al., Mol. Cell Biol. 7:887-897 (1987); Dzierzak et al., Nature 331:35-41 (1988); Karlsson et al., Proc. Natl. Acad. Sci. USA 78:3629-3633 (1988); Miller et al., J. Virol., 62:4337-4345 (1988); Bender et al., Mol. Cell. Biol., 9:1426-1434 (1989)). The discovery of major hypersensitive sites (HS) far upstream of the human .beta.-globin gene locus, constituting the .beta.-Locus Control region (.beta.-LCR), has given new hope for successful gene therapy of human .beta.-globin gene disorders. (Tuan and London, Proc. Natl. Acad. Sci. USA 81:2718-2722 (1984); Tuan et al., Proc. Natl. Acad. Sci. USA 82:6384-6388 (1985); Forrester et al., Proc. Natl. Acad. Sci. USA 89:1968-1972 (1986); Grosveld et al., Cell 51:975-985 (1987)). LCR derivatives are able to confer erythroid-specific, high, integration site-independent expression of a linked .beta.-globin gene in transgenic mice and murine erythroleukemia (MEL) cells, which mimic adult erythroid differentiation (Grosveld et al., Cell 51:975-985 (1987)). Because the activity of each HS site has now been localized to small DNA fragments (U.S. Pat. No. 5,126,260; Curtin et al., Proc. Natl. Acad. Sci. USA 86:7082-7086 (1989); Forrester et al., Proc. Natl. Acad. Sci. USA 86:5439-5443 (1989); Ryan et al., Genes Dev. 3:314-323 (1989); Tuan et al., Proc. Natl. Acad. Sci. USA, 86:2554-2558 (1989); Collis et al., EMBO J., 9:233-240 (1990); Ney et al., Genes Dev. 4:993-1006 (1990); Philipsen et al., EMBO J., 9:2159-2167 (1990); Talbot et al., EMBO J., 9:2169-2178 (1990); Pruzina et al., Nucleic Acids Res., 19:1413-1419 (1991); Walters et al., Nucleic Acids Res., 19:5285-5393 (1991)), it has become possible to construct retroviral vectors transducing .beta.-LCR derivatives linked to the human .beta.-globin gene and its proximal cis-acting elements (Novak et al., Proc. Natl. Acad. Sci., USA, 87:3386-3390 (1990); Chang et al., Proc. Natl. Acad. Sci. USA, 89:3107-3110 (1992)). However, these [.beta.-globin/LCR] retroviruses have low titer, are very unstable with multiple rearrangements upon transmission of the proviral structure, and provide a relatively modest and highly variable enhancement of .beta.-globin gene expression in infected murine erythroleukemia (MEL) cells (Novak et al., Proc. Natl. Acad. Sci. USA 87:3386-3390 (1990); Chang et al., Proc. Natl. Acad. Sci. USA 89:3107-3110 (1992)).
U.S. Pat. No. 5,126,260 describes DNAaseI hypersensitive sites that constitute the .beta.-LCR and, in particular, identifies the HS2 enhancer within the .beta.-LCR structure. U.S. Pat. No. 5,126,260 also claims the use of .beta.-LCR and HS2 derivatives in gene transfer protocols, including retrovirus-mediated gene transfer, to obtain high expression level of the human .beta.-globin gene. However, U.S. Pat. No. 5,126,260 does not identify specific means by which stable proviral transmission of [.beta.-globin/LCR] retroviruses can be achieved.
It is therefore an object of the present invention to provide retroviral vectors for stable transduction of the .beta.-globin gene and .beta.-locus control region derivatives and other erythroid specific genes.